Focused evolution of HIV-1 neutralizing antibodies revealed by structures and deep sequencing

Abstract

Antibody VRC01 is a human immunoglobulin that neutralizes about 90% of HIV-1 isolates. To understand how such broadly neutralizing antibodies develop, we used x-ray crystallography and 454 pyrosequencing to characterize additional VRC01-like antibodies from HIV-1-infected individuals. Crystal structures revealed a convergent mode of binding for diverse antibodies to the same CD4-binding-site epitope. A functional genomics analysis of expressed heavy and light chains revealed common pathways of antibody-heavy chain maturation, confined to the IGHV1-2*02 lineage, involving dozens of somatic changes, and capable of pairing with different light chains. Broadly neutralizing HIV-1 immunity associated with VRC01-like antibodies thus involves the evolution of antibodies to a highly affinity-matured state required to recognize an invariant viral structure, with lineages defined from thousands of sequences providing a genetic roadmap of their development.

Fig. 1

Identification and characterization of mAbs from HIV-1– infected donors 74 and 0219. (A) RSC3 analysis of serum. Twelve sera from the IAVI Protocol G cohort and one serum from the CHAVI 001 cohort (donor 0219) were analyzed for RSC3 reduction in serum neutralization on HIV-1 strains JR-FL, PVO.4, YU2, and ZA12.29. Blue bars show the mean serum reduction in neutralization IC₅₀ resulting from RSC3 versus ΔRSC3 competition. Sera with the greatest reduction were further analyzed on HIV-1 strains Q168.a2, RW020.2, Du156.12, and ZM109.4. Red bars show the mean reduction on eight viruses. (B) Flow cytometric identification of RSC3-reactive IgG⁺ B cells from donors 74 and 0219. Gating and percentage of IgG⁺ B cells of interest (RSC3⁺ΔRSC3^–) are indicated, with 40 and 26 sorted single B cells from donors 74 and 0219, respectively. Additional sorting details are shown in fig. S1. (C) Protein sequences of heavy- and light-chain variable regions of mAbs VRC-PG04 and VRC-PG04b, isolated from donor 74, and mAbs VRC-CH30-34 isolated from donor 0219. Sequences are aligned to the putative germline ancestral genes and to previously identified broadly neutralizing antibodies VRC01, VRC02, and VRC03. Framework regions (FR) and complementarity-determining regions (CDRs) are based on Kabat nomenclature (46). (D) Neutralization dendrograms. VRC-PG04 and VRC-CH31 were tested against genetically diverse Env-pseudoviruses representing the major HIV-1 clades. Neighbor-joining trees display the protein distance of gp160 sequences from 178 HIV-1 isolates tested against VRC-PG04 and a subset (80 isolates) tested against VRC-CH31. A scale bar denotes 1% distance in amino acid sequence. Tree branches are colored by the neutralization potencies of VRC-PG04 and VRC-CH31 against each particular virus.

Fig. 2

Structure of antibodies VRC-PG04 and VRC03 in complex with HIV-1 gp120. (A) Overall structures. The liganded complex for the Fab of antibody VRC-PG04 from donor 74 and the HIV-1 gp120 envelope glycoprotein from isolate 93TH057 is depicted with polypeptide backbones in ribbon representation in the left image. The complex of Fab VRC03 from donor 45 is depicted in the right image, with surfaces of all variable domain residues that differ between VRC03 and VRC-PG04 colored according to their chemical characteristics. (B and C) Interaction close-ups. Critical interactions are shown between the CD4-binding loop of gp120 (purple) and the CDR H2 region of the broadly neutralizing mAbs VRC03 and VRC-PG04 (reported here) and VRC01 [reported previously (19)], with hydrogen bonds depicted as dotted lines. The 1.9 and 2.1 Å resolution structures of VRC03 and VRC-PG04, respectively, were sufficient to define interfacial waters shown in (C), which were unclear in the 2.9 Å structure of VRC01. The orientation shown in (C) is ~180° rotated about the vertical axis from the orientation shown in (B).

Fig. 3

Focused evolution of VRC01-like antibodies. (A) Antibody convergence. The gp120 portions of liganded complexes with VRC01, VRC03, and VRC-PG04 were superimposed to determine the average antibody per residue Cα deviation, and the per residue hydrophobic interaction (ΔⁱG) was calculated (47). These two quantities were found to correlate (P = 0.0427), with antibody residues containing strong hydrophobic interactions (e.g., at positions 53 and 55 in the heavy chain, and 91 and 97 in the light chain, VRC-PG04–relative numbering) displaying high structural conservation. This correlation is visualized on VRC-PG04 in the left image, where the ribbon thickness is proportional to the corresponding per residue Cα deviation and the paratope surface is colored according to hydrophobicity, from white (low) to red (high); notably, red surface patches map to thin ribbons. (B) Epitope convergence. The HIV-1 gp120 surface involved with CD4 binding contains conformationally invariant regions (e.g., associated with the outer domain) and conformationally variable regions (e.g., associated with the bridging sheet). We previously hypothesized that the conformationally invariant outer domain contact for CD4 represents a site of vulnerability (19). We analyzed the precision of CD4-binding-site ligand recognition (vertical axis) versus the IC₈₀ neutralization breadth (horizontal axis) and observed significant correlation (R² = 0.6, P = 0.040). (C) Divergences in sequence and convergences in recognition. The development of VRC01-like antibodies involves a heavy chain derived from the IGHV1-2*02 allele and selected light-chain Vκ alleles. The far left image depicts a ribbon representation model of a putative germline antibody. Somatic hypermutation during the process of affinity maturation leads to a divergence in sequence, yet results in the convergent recognition of similar epitopes. Intersection of the epitope surfaces recognized by VRC01, VRC03, and VRC-PG04 (far right image), reveals a notable similarity to the site of vulnerability. The primary divergence of this intersection from the hypothesized site of vulnerability occurs in the region of HIV-1 gp120 recognized by the light chain of the VRC01-like antibodies. Although the separate epitopes on gp120 do show differences in recognition surface, these primarily involve the bridging sheet region, which is likely to adopt a different conformation in the functional viral spike before engagement of CD4.

Fig. 4

Deep sequencing of expressed heavy and light chains from donors 45 and 74. (A) Heavy- and light-chain complementation. The neutralization profiles of VRC01 and VRC03 (donor 45), VRC-PG04 (donor 74), and VRC-CH31 (donor 0219) and their heavy- and light-chain chimeric swaps are depicted with 20-isolate neutralization dendrograms. Explicit neutralization IC₅₀s are provided in table S13. (B) The repertoire of heavy-chain sequences from donor 45 (2008 sample) and donor 74 (2008 sample). Heavy-chain sequences are plotted as a function of sequence identity to the heavy chain of VRC01 (left), VRC03 (middle), and VRC-PG04 (right) and of sequence divergence from putative genomic V_H alleles: Upper row plots show sequences of putative IGHV1-2*02 allelic origin; lower row plots show sequences from other allelic origins. Color coding indicates the number of sequences. (C) Repertoire of expressed light-chain sequences from donor 45 (2001 sample). Light-chain sequences are plotted as a function of sequence identify to VRC01 (left) and VRC03 (right) light chains, and of sequence divergence from putative genomic V-gene alleles. Sequences with two-residue deletions in the CDR L1 region (which is observed in VRC01 and VRC03) are shown as black dots. Two light-chain sequences, with 92.0% identity to VRC01 (sequence ID 181371) and with 90.3% identity to VRC03 (sequence ID 223454) are highlighted with red triangles. (D) Functional assessment of light-chain sequences identified by deep sequencing. The neutralization profiles of sequence 181371 reconstituted with the VRC01 heavy chain (named gVRC-L1_d45) and of sequence 223454 reconstituted with the VRC03 heavy chain (named gVRC-L2_d45) are depicted with 20-isolate neutralization dendrograms; explicit neutralization IC₅₀s are shown in table S22. (E) Functional assessment of heavy-chain sequences identified by deep sequencing. Heavy-chain sequences from donors 45 and 74 were synthesized and expressed with either the light chain of VRC01 or VRC03 (for donor 45) or the light chain of VRC-PG04 (for donor 74) and evaluated for neutralization. Neutralizing sequences are shown as red stars and are labeled. gVRC-H(n)_d74 refers to the heavy chains with confirmed neutralization when reconstituted with the light chain of VRC-PG04, with controls as described in (34).

Fig. 5

Maturational similarities of VRC01-like antibodies in different donors revealed by cross-donor phylogenetic analysis. (A) Maximum-likelihood trees of heavy-chain sequences of the IGHV1-2*02 origin from donor 45 (left) and donor 74 (right). The subset of sequences shown was selected based on the germline divergence as described in (23). The donor 45 tree is rooted by the putative reverted unmutated ancestor of the heavy chain of VRC01 and also includes specific neutralizing sequences from donors 74 and 0219 (shown in red). Similarly, the donor 74 tree is rooted in the putative reverted unmutated ancestor of the heavy chain of VRC-PG04, and sequences from donors 45 and 0219 are included in the cross-donor phylogenetic analysis. Bars representing 0.1 changes per nucleotide site are shown. Insets show J chain assignments for all sequences within the neutralizing subtree identified by an iterative neighbor-joining tree analysis as described in (23). (B) Phylogenetically inferred maturation intermediates. Backbone ribbon representations are shown for HIV-1 gp120 (red) and the heavy-chain variable domains (green). Critical intermediates inferred from the phylogenetic tree in (A) are labeled I_d45, II_d45, III_d45, I_d74, and II_d74. The number of V_H-gene mutations is provided (e.g., for the 23 mutations associated with the first intermediation of donor 45, “I_d45: 23”), and the location of these is highlighted in the surface representation and colored according to their chemistry.

Fig. 6

Analysis of the heavy-chain antibodyome of donor 74 and identification of heavy chains with HIV-1 neutralizing activity. Identity/divergence-grid analysis, cross-donor phylogenetic analysis, and CDR H3 analysis were coupled to functional characterization of selected heavy-chain sequences. This provides a means for identification of novel heavy chains with HIV-1 neutralizing activity. (A) Identity/divergence-grid analysis. The location of the 63 synthesized IGHV1-2*02 heavy chains from donor 74 is shown, including neutralizing (red stars) and non-neutralizing (black stars) sequences. (B) Cross-donor phylogenetic analysis and CDR H3 lineage analysis. A maximum-likelihood tree of the 70 synthesized heavy-chain sequences (including 7 non-IGHV1-2*02 sequences) is rooted at the putative reverted unmutated ancestor of VRC-PG04. The probe-identified VRC-PG and VRC-CH antibodies are shown in red text along with the 24 genomically identified heavy-chain sequences, gVRC-H(1-24)_d74, which were found to neutralize HIV-1 when reconstituted with the light chain of VRC-PG04. Grid locations and CDR H3 classes are specified for neutralizing and non-neutralizing sequences. Within each CDR H3 class, all sequences with identical CDR H3s are highlighted in orange in the far right grids (with the number of total sequences corresponding to each CDR H3 class shown). (C) Expression levels of selected heavy chains reconstituted with the light chain of VRC-PG04 versus breadth of neutralization. (D) Neutralization potency of reconstituted cross-donor phylogeny-predicted antibodies on seven HIV-1 isolates. (E) CDR H3 analysis of donor 74 heavy-chain sequences. For each of the 110,386 sequences derived from the IGHV1-2*02 allele, the CDR H3 was determined, and its percent identity to that of the VRC-PG04 heavy chain was color coded as shown and graphed. The sequences with high CDR H3 identity to VRC-PG04 reside in regions of high overall heavy-chain sequence identity, even for sequences with a low divergence from IGHV1-2*02.